The goal of this project is to provide a comprehensive description of human subtelomeric DNA structure. Human subtelomeric DNA regions are characteristically very GC-rich (and gene-rich), contain large stretches of duplicated and low-copy DNA, and possess remarkable large-scale variations in sequence content at many telomeres on the order of hundreds of kilobases between alleles of homologous chromosomes. These regions are involved in telomerase-independent recombination pathways of telomere maintenance/replication, and are somatically and evolutionarily plastic Telomere-associated position effects upon subtelomeric gene expression can extend over large subtelomeric regions, and are affected by the content and organization of particular subtelomeric repeat elements. Global detailed analyses of subtelomeric structure and variation are therefore very likely to reveal important clues regarding mechanisms of genome replication, chromosome stability, and subtelomeric gene function and evolution. The atypical sequence organization and large-scale allelic variation of many subtelomeric regions are likely to complicate the telomeric closure phase of human genome reference sequence completion. My lab has taken a focused approach to subtelomeric DNA analysis over the past 8 years of this project generating and mapping clones, DNA probes and sequence islands that collectively span most of the subtelomeric regions from the distal-most (TTAGGG) n repeats at the molecular ends of every chromosome to extended regions of chromosome-specific DNA characteristic of individual chromosome arms. The proposed experiments are designed to integrate human genome sequence data emerging from production sequencing facilities with telomeric mapping and sequencing data generated in our lab in order to facilitate telomeric reference sequence closure, and to use both physical mapping methods and computational comparisons of sequence data to analyze and annotate subtelomeric sequence content and organization in the context of the reference sequence.